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961.
Chang W Pratt S Chen TH Bourguignon L Shoback D 《The Journal of biological chemistry》2001,276(47):44129-44136
The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation. 相似文献
962.
Mäkiniemi M Hillukkala T Tuusa J Reini K Vaara M Huang D Pospiech H Majuri I Westerling T Mäkelä TP Syväoja JE 《The Journal of biological chemistry》2001,276(32):30399-30406
Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions. 相似文献
963.
Iida J Pei D Kang T Simpson MA Herlyn M Furcht LT McCarthy JB 《The Journal of biological chemistry》2001,276(22):18786-18794
Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion. In this study, we sought to identify components of the cell surface of a vertical growth phase melanoma cell line, WM1341D, that mediate invasive cellular behavior. We determined by antisense inhibition that melanoma chondroitin sulfate proteoglycan (MCSP) and membrane-type 3 matrix metalloproteinase (MT3-MMP) expressed on WM1341D are required for invasion of type I collagen and degradation of type I gelatin. MT3-MMP co-immunoprecipitated with MCSP in WM1341D melanoma cells cultured on type I collagen or laminin. The association between MT3-MMP and MCSP was largely disrupted by removing chondroitin sulfate glycosaminoglycan (CS) from the cell surface, suggesting CS could mediate the association between the two cell surface core proteins. Recombinant MT3-MMP and MT3-MMP from whole cell lysates of WM1341D cells were specifically eluted from CS- conjugated affinity columns. The results indicate that MT3-MMP possesses the potential to promote melanoma invasion and proteolysis and that the formation of a complex between MT3-MMP and MCSP may be a crucial step in activating these processes. 相似文献
964.
Shisheva A Rusin B Ikonomov OC DeMarco C Sbrissa D 《The Journal of biological chemistry》2001,276(15):11859-11869
The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P(2), thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve products' formation, we have examined the localization of PIKfyve protein versus enzymatic activity and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol ( approximately 76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal elements) ( approximately 20%), and plasma membrane (( approximately )4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption, and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve's role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action. 相似文献
965.
Weinman EJ Evangelista CM Steplock D Liu MZ Shenolikar S Bernardo A 《The Journal of biological chemistry》2001,276(45):42339-42346
Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets. 相似文献
966.
967.
The lysine-rich C-terminal tail of heparin affin regulatory peptide is required for mitogenic and tumor formation activities 总被引:3,自引:0,他引:3
Bernard-Pierrot I Delbe J Caruelle D Barritault D Courty J Milhiet PE 《The Journal of biological chemistry》2001,276(15):12228-12234
Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two beta-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic. 相似文献
968.
969.
970.
Arrecubieta C Hammarton TC Barrett B Chareonsudjai S Hodson N Rainey D Roberts IS 《The Journal of biological chemistry》2001,276(6):4245-4250
The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process. 相似文献